BACKGROUND: The purpose of this study was to evaluate (1) the washout kinetics of 99mTc-labeled tetrofosmin, separately for myocardium with normal and reduced perfusion, and (2) its influence on quantitative analysis in a 1-day stress-rest protocol. METHODS AND RESULTS: Twenty-five patients with angiographically proved coronary artery disease underwent bicycle exercise stress testing with injection of 200 MBq 99mTc-labeled tetrofosmin and first single-photon emission computed tomographic (SPECT) imaging 40 minutes after injection. A second SPECT was acquired 2.3 +/- 0.4 hours after the first one immediately before rest injection of 800 MBq 99mTc-labeled tetrofosmin. The rest (third) SPECT was acquired 15 minutes thereafter. The relative washout fraction per time (WOFt) was calculated assuming linear washout kinetics. Thirty-three regional uptake values per study were calculated, normalized to the perfusion maximum (100%) in either the postexercise SPECT and the rest SPECT, for the latter with and without correction of remaining counts from stress injection. In regions with normal perfusion, WOFt was 11.5% +/- 3.5% per hour. In regions with markedly reduced perfusion (relative uptake < 50%, WOFt was 8.3% +/- 9.9% per hour. The highest variation of the relative uptake values between rest SPECT with and without correction of remaining counts from stress injection was 5.4% +/- 3.5% in regions with stress-induced ischemia. CONCLUSION: To use a 1-day protocol with a stress-rest radioactivity ratio of 1:4 and an interval of more than 2 hours between the examinations, a correction for remaining counts from stress injection seems not to be necessary for the quantitative analysis of rest SPECT.